Ubiquitination as a priming process of PKC alpha and PKC epsilon degradation in the alphaT3-1 gonadotrope cell line.
OBJECTIVE
Protein kinase C (PKC) is a family of isoenzymes playing a key role in the regulation of gonadotrope cell functions. Specific PKC isoforms are activated and downregulated differentially by gonadotropin-releasing hormone (GnRH) and the phorbol ester TPA. In the present study, focusing mainly on PKC epsilon, the mechanisms underlying the proteasome-dependent downregulation of GnRH-activated PKC epsilon and TPA-sensitive PKC alpha and epsilon isoenzymes were investigated in alphaT3-1 gonadotrope cells.


RESULTS
In pull-down assays involving the use of glutathione-agarose affinity beads conjugated with a GST-fusion protein containing ubiquitin-associated domains of Rad23 that bind very likely to K48-linked polyubiquitinated proteins, TPA induced rapid (within 15 min) and sustained (up to 4 h) PKC alpha and PKC epsilon polyubiquitination. However, GnRH selectively elicited receptor-dependent polyubiquitination of PKC epsilon, but not that of PKC alpha. The GnRH-evoked PKC epsilon polyubiquitination was a strong, fast process (taking place as early as 10 min) which decreased progressively with time (but was still detectable after 4 h of treatment). In addition, no apparent association between PKC epsilon and the lysosomal compartment was observed upon performing double-labeling immunofluorescence and confocal microscopy, after either 10 min or 1 hour of stimulation by GnRH or the phorbol ester.


CONCLUSIONS
In alphaT3-1 gonadotrope cells, polyubiquitination is therefore the event triggering GnRH-evoked PKC epsilon desensitization as well as TPA-induced PKC alpha and PKC epsilon downregulations; it precedes the respective isoenzyme's degradation by the proteasome complex.
Poulin
Benoit
B
CNRS UMR 6231, Université de la Méditerranée, Faculté de Médecine, Marseille, France.
Maccario
Hélène
H
Thirion
Sylvie
S
Junoy
Brice
B
Boyer
Bénédicte
B
Enjalbert
Alain
A
Drouva
Sophia V
SV
D000818:Animals; D002460:Cell Line; D015536:Down-Regulation / Q000187:drug effects; D052681:Gonadotrophs / Q000166:cytology / Q000378:metabolism* / Q000502:physiology*; D007987:Gonadotropin-Releasing Hormone / Q000031:analogs & derivatives / Q000037:antagonists & inhibitors / Q000494:pharmacology; D007527:Isoenzymes / Q000378:metabolism; D051379:Mice; D019319:Okadaic Acid / Q000494:pharmacology; D009842:Oligopeptides / Q000494:pharmacology; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D051571:Protein Kinase C-alpha / Q000378:metabolism*; D051744:Protein Kinase C-epsilon / Q000378:metabolism*; D021381:Protein Transport; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D054875:Ubiquitination / Q000502:physiology*
D007527:Isoenzymes; D009842:Oligopeptides; C090535:benzyloxycarbonyl-isoleucyl-glutamyl(O-tert-butyl)-alanyl-leucinal; C541922:pyroglutamyl-histidyl-tryptophyl-seryl-tyrosyl-alanyl-leucyl-arginyl-N-ethylprolinamide; D019319:Okadaic Acid; D007987:Gonadotropin-Releasing Hormone; C057022:iturelix; D051571:Protein Kinase C-alpha; D051744:Protein Kinase C-epsilon; D046988:Proteasome Endopeptidase Complex; D013755:Tetradecanoylphorbol Acetate